Centrosomes, which consist of a pair of centrioles wrapped within a cloud of pericentriolar material (PCM), are the primary microtubule-organizing centers (MTOCs) in most cells. In Caenorhabditis elegans, the kinase ZYG-1 is essential for the replication of centrosomes. Embryos lacking maternal ZYG-1 activity fail to replicate the paternally contributed centriole pair, and are thus unable to form bipolar spindles following first division. In contrast, loss of paternal ZYG-1 activity results in duplication failure during male meiosis, and the production of sperm with a single centriole. These sperm can still fertilize eggs but the resulting embryos assemble a monopolar rather than bipolar spindle at first division. Several recently published reports indicated that centrosome duplication might be regulated differently in different tissues. To investigate this in C. elegans, we have been analyzing the effects of zyg-1 mutations on centrosome duplication in the male and female germ lines. We have found that small truncations of the c-terminus of ZYG-1 cause a novel phenotype: the production of embryos with supernumerary replication-incompetent centrosomes. These embryos inherit up to eight centrioles that are unable to duplicate. We have found the extra centrioles arise solely from defects in the male germ line and that this phenotype behaves as a gain of function. In contrast, failure of mutant embryos to duplicate these centrosomes is due to a loss of function of maternal ZYG-1. Thus, a single mutation behaves as a loss-of-function allele in one developmental context and a gain-of-function allele in another, suggesting that ZYG-1 is subject to different forms of regulation in different tissues. Consistent with this, we find that these mutant forms of ZYG-1 localize normally to meiotic centrosomes but are almost absent from mitotic centrosomes. The low level of mutant ZYG-1 at mitotic centrosomes is not the result of an unstable protein, as the truncated forms of ZYG-1 are expressed at normal levels. To investigate the centrosome amplification phenotype further, we characterized centrosome behavior during meiosis in live and fixed spermatocytes. We found that centrosomes behave normally through the end of meiosis I but rapidly amplified as cells approached meiosis II. Amplification of centrosomes depends upon the centrosome duplication factor SAS-6 and additional genetic analysis indicates that most, if not all, of the extra centrosomes produced in the male germ line are structurally intact. We have confirmed this using transmission electron microscopy. In summary, our data indicate that centrosome duplication is regulated differently in mitosis and meiosis. To understand these differences at a molecular level, we are working to identify factors that provide mitotic or meiotic specific regulation of centrosome duplication. In a parallel study, we are also investigating how SAS-6 might be subject to meiosis-specific regulation. SAS-6 is a coiled-coil protein that specifies the nine-fold symmetric architecture of the centriole. We have recently obtained an unusual allele of sas-6 in which meiotic but not mitotic centriole duplication appears perturbed. This suggests that SAS-6, like ZYG-1 is regulated differently during the two modes of division. We plan to conduct in depth genetic and cytological analysis to characterize this defect. If we find that this mutation conveys a strict meiotic defect, we will then carry out a genetic suppressor screen (as has successfully been done for zyg-1) to identify genetic elements that specifically regulate meiotic centriole duplication. Finally we have undertaken a genetic screen to identify new alleles of sas-6 that hopefully will reveal novel features of its regulation. Our screening strategy involves identifying mutations that fail to complement a sas-6 deletion allele. Of 74 initial candidates we identified, 38 have been eliminated upon rescreening while two meet the criteria for sas-6 alleles. These two candidates, along with any others that test positive, will be analyzed by sequencing to determine if the sas-6 gene carries a mutation. New sas-6 alleles will then be characterized using established genetic and cytological methods. Ultimately, we hope to identify domains of the protein that are important for centriole architecture and for regulating SAS-6 activity in a cell- and tissue-specific manner.